Expansions of CTG/CAG repeats at specific sites in the human genome cause a number of neurological diseases, including myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. These diseases arise when the trinucleotide (triplet) repeat expands beyond a threshold of about 25-35 repeats to a length that has pathologic consequences. This application focuses on the basis for CTG/CAG repeat instability. Studies in model systems have shown that virtually any process that exposes single strands of DNA-transcription, replication, repair, recombination-can destabilize triplet repeats. CTG and CAG repeats in single strands form hairpins, which are thought to be the key intermediate in repeat expansion. Hairpins either interfere with normal repair or trigger aberrant repair, which leads to expansion of the repeat. In no case, however, has the pathway for expansion of triplet repeats in humans been defined. Using a novel direct-selection assay, we have identified transcription and genome-wide demethylation as two processes that substantially destabilize triplet repeats in mammalian cells. Transcription-induced instability may be a major contributor to the somatic diversity that accumulates with age in patient tissues, especially in those such as neurons that no longer divide. Demethylation-induced repeat instability is directly relevant to the germline events that lead to the progressive worsening of the disease phenotype in subsequent generations-the clinical phenomenon of anticipation. We propose to define the specific proteins responsible for transcription- and demethylation-induced instability, and in that way to define the molecular mechanisms underlying CTG/CAG repeat instability. We will use siRNA and dominant-negative mutants in conjunction with our direct-selection assays in human cells. We will extend our studies to mice to examine the effects of altered genomic methylation on repeat stability in the germline. Finally, we propose to use our direct-selection assay to screen insertion vector libraries, siRNA libraries, and chemical libraries to identify genes that alter repeat stability. Our goal is to delineate those processes that are responsible for both the germline and somatic CTG/CAG repeat instability that characterizes myotonic dystrophy and other neurological diseases. [unreadable] [unreadable]